Platelet transfusions are a life-saving treatment for health conditions related to cancer therapy, bone marrow transplantation, bone marrow failure, sepsis, genetic platelet disorders, and some autoimmune conditions. Currently, over 2 million apheresis platelet units and over 4 million pooled platelet concentrate units are transfused in the U.S. each year. However the limited shelf life of donor platelets leads to frequent shortages. Moreover, multiply transfused patients often become refractory due to allosensitization. The advent of human embryonic stem (hES) and induced pluripotent stem cell (hIPSC) technology has raised the possibility of producing platelets in vitro for clinical use. This would allow for a essentially limitless supply of on-demand platelets and could be engineered to overcome allosensitization problems. However, current protocols are hampered by low platelet production efficiency. The major bottleneck is at the level of terminal megakaryocyte (Mk) maturation and platelet release, which is about 1000-fold lower in vitro compared to primary Mks in vivo. The major objective of this proposal is to further understand the physiologic mechanisms that regulate terminal thrombopoiesis and to apply these to enhance hIPSC in vitro platelet production. Terminal Mk maturation requires the action of a small group of key transcription factors (RUNX1, GATA1, Friend of GATA1, and FLI1) that physically associate with one another during differentiation. This drives high- level activation or repression of genes involved in polyploidization, platelet membrane genesis, platelet constituent biosynthesis and proplatelet formation. We hypothesize that key signaling events control the assembly of this Mk enhancesome complex, and can be exploited to enhance IPSC based platelet production. We have previously identified key signaling events that regulate differentiation-dependent interaction among these factors. This includes src-family kinase (SFK) mediated tyrosine phosphorylation of RUNX1 and serine phosphorylation of FLI1 at codon 10. These findings are supported by work from other groups showing marked enhancement of primary murine and human Mk maturation by SFK inhibitors. The aims of this proposal are to further elaborate these signaling pathways and to determine the extent to which their pharmacologic and/or genetic manipulation enhances hIPSC-derived Mk maturation and platelet release, while preserving platelet function. In addition, we propose to apply the newly developed technique Fluorescent in situ RNA seq (FISSEQ) for non-biased discovery of additional signaling pathways that are engaged when Mk progenitors physically interact with the vascular sinusoidal niche, the physiologic site of terminal Mk maturation and platelet release. The expected outcome of this project is the elucidation of specific physiologic signaling pathways that can be manipulated to increase hIPSC platelet production efficiency.